Project Summary Scalable genetically-encoded technologies that enable the construction of systems that receive, process, and transmit molecular information are essential to advancing basic biological research, applied biomedical research, and biotechnology. Such enabling technologies will significantly advance our ability to monitor, interface with, and program the dynamic cellular state. An interdisciplinary approach spanning the areas of engineering, molecular biology, and biochemistry will be applied to develop modular and programmable RNA-based platforms to build user-defined sensing-actuation systems that will translate a molecular input into a change in cellular function. By coupling RNA structure-function relationships, engineering design strategies, and computational modeling tools, frameworks will be developed for constructing ligand-controlled RNA regulatory systems that will be utilized to respond to and manipulate molecular information in living systems. Our goals are to develop the experimental and computational frameworks for the reliable construction and programming of integrated RNA-based control systems that exhibit user-defined regulatory properties. These systems will be utilized to advance our understanding of effective strategies for integrating functional network and control system architectures. Specific aims are to: 1. Develop a trans-ribozyme switch platform. This will include: (i) developing a trans-ribozyme platform that exhibits biologically relevant cleavage efficiencies and binding affinities; (ii) developing an associated trans-ribozyme expression system for effective regulation of target protein levels in vivo; (iii) developing a trans-ribozyme switch platform. 2. Develop computational tools for programming switch properties. This will include: developing (i) computational models of ligand-controlled RNA regulatory systems to predict parameters critical to system response, (ii) an in silico framework for the prediction of in vivo folding parameters from sequence, (iii) a sequence-to-function framework for the forward design of switch properties. 3. Develop cell-based screens and selections for new switch functions. This will include: developing cell-based (i) screening and (ii) selection strategies for new ribozyme switches; (iii) implementing these strategies for the generation of ribozyme switches with new sensory or response properties. 4. Examine the effects of introducing controlled perturbations to the yeast pheromone-responsive MAPK pathway. This will include: examining the effects of (i) titrating MAPK proteins with cis- ribozyme switches and (ii) introducing controlled perturbations to the MAPK pathway with trans- ribozyme switches on signaling through the pathway and system response.